The transforming human gammaherpesviruses EBV and KSHV establish stable latent infections in B cells, providing a lifelong reservoir of virus tha can contribute to the development of malignant disease. Thus, defining the mechanisms that govern long-term latency in B cells is critical for designing rational strategies to prevent diseas. In vivo studies of gammaherpesviruses in humans have been severely limited by the difficulties of working in the natural host. Murine gammaherpesvirus 68 (MHV68) is related to EBV and KSHV and causes lymphomas and lymphoproliferative disease in mice, providing a readily manipulable small animal model for mechanistic studies of the virus/host relationship in vivo. Like EBV and KSHV, MHV68 expresses a defined set of miRNAs whose functions during infection are largely unknown. As these virus-encoded miRNAs are abundantly expressed in vivo in B cells during long-term latency and in hyperplastic B cell lesions during lymphoproliferative disease, we hypothesize that these viral miRNAs play key roles in latency and pathogenesis. To test this hypothesis, we will 1) Determine the functional role of individual MHV68 miRNAs in long-term latency and lymphoproliferative disease; 2) Identify specific virus and host targets of the key miRNAs using cutting-edge CLIP and deep sequencing approaches coupled with in vitro and in vivo validation; 3) Define the in vivo biological relevance of target genes and determine their roles in B cell maturation and survival. We anticipate that there will be significant overlap between the mRNA targets and cellular pathways regulated by MHV68, KSHV, and EBV miRNAs. The systematic in vivo analyses of MHV68 miRNA mutants, in conjunction with HITS-CLIP technology and in vivo validation of miRNA targets, provides an extremely powerful means to determine the molecular mechanism by which miRNAs contribute to gammaherpesvirus infection in vivo, and should allow us to define the contribution of repression of these shared targets to gammaherpesvirus latency and pathogenesis.